Design of Novel Amphipathic α-Helical Antimicrobial Peptides with No Toxicity as Therapeutics against the Antibiotic-Resistant Gram-Negative Bacterial Pathogen, Acinetobacter Baumannii

We designed de novo and synthesized two series of five 26-residue amphipathic α-helical cationic antimicrobial peptides (AMPs) with five or six positively charged residues (D-Lys, L-Dab (2,4-diaminobutyric acid) or L-Dap (2,3-diaminopropionic acid)) on the polar face where all other residues are in the D-conformation. Hemolytic activity against human red blood cells was determined using the most stringent conditions for the hemolysis assay, 18h at 37°C, 1% human erythrocytes and peptide concentrations up to 1000 μg/mL (~380 μM). Antimicrobial activity was determined against 7 Acinetobacter baumannii strains, resistant to polymyxin B and colistin (antibiotics of last resort) to show the effect of positively charged residues in two different locations on the polar face (positions 3, 7, 11, 18, 22 and 26 versus positions 3, 7, 14, 15, 22 and 26). All 10 peptides had two D-Lys residues in the center of the non-polar face as “specificity determinants” at positions 13 and 16 which provide specificity for prokaryotic cells over eukaryotic cells. Specificity determinants also maintain excellent antimicrobial activity in the presence of human sera. This study shows that the location and type of positively charged residue (Dab and Dap) on the polar face are critical to obtain the best therapeutic indices.


Introduction
The growing emergence of pathogenic bacteria with clinically significant resistance to conventional antibiotics is a major public health concern [1][2][3][4][5]. As noted by Falanga, et al. [6] we are facing a worldwide re-emergence of infectious diseases and a rapid increase in multidrug-resistant (MDR) bacteria, threatening the world with a return to the pre-antibiotic era. Indeed, there are now "Superbugs" that are resistant to most or all available antibiotics [7]. The scope of the challenge in tackling drug-resistant infections globally is reported in detail in a 2016 review on antimicrobial resistance [4]. Thus, it was estimated that, by 2050, 10 million lives a year will be at risk due to the rise of drug-resistant infections if proactive solutions are not quickly found to slow the rate of drug resistance. At present, 700,000 people die every year from drug resistant strains of common bacterial infections, HIV, TB and malaria [4]. While the 2016 review [4] offered a plethora of approaches to slowing down or preventing future bacterial resistance to antibiotics (e.g., promoting vaccine use, avoiding unnecessary antibiotic use, better water and sanitation, decrease in environmental pollution), the fact remains that organisms resistant to conventional antibiotics will still be present and must be dealt with. Indeed, it has frequently been asserted that, as part of a global response to MDR bacteria, we must increase the number of effective antimicrobial drugs to defeat infections that have become resistant to existing antibiotics [4]. Unfortunately, antibiotic discovery has stalled just as we need it the most. Between 1929 and the 1970s, more than 20 new classes (not just analogs of an existing class) of antibiotic reached the market [3]. Since then, only two new classes have reached the market, with the worldwide antibiotic pipeline for new antibiotic classes active against highly resistant Gram-negative bacteria being almost non-existent [3]. It is estimated that only 4 new classes of antibiotics can be expected in the next 30 years, while antibiotic resistance to some pathogens may more than double in the same period [4]. Although, in the 1970s and 1980s, the pharmaceutical industry did produce a stream of antibiotics, these were not new classes but analogs of existing classes [3]. The fundamental problem with this approach is that, although analog development is low risk compared to novel class discovery and development, analogs eventually became more difficult to come by and the process hits a dead end.
A potential solution to the crisis of medically resistant strains of bacteria lies in a ubiquitous response in nature to bacterial infections, namely the production of antimicrobial peptides (AMPs) [6,[8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24]. AMPs are produced by a wide variety of organisms, including bacteria, fungi, plants, insects, amphibians, crustaceans, fish and mammals (including humans) [25]. AMPs (specifically, cationic AMPs) are fast-acting bactericides with generally broad spectrum activity [25]. In addition, AMPs in general do not have specific targets (unlike traditional antibiotics), their mode of action generally involving nonspecific interactions with the cytoplasmic membrane of bacteria. This causes peptide accumulation in the membrane, leading to increased permeability and loss of barrier function [8,9,12,13,[15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30]. Development of resistance is not expected since this would require substantial changes in the lipid composition of the cell membranes of microorganisms. The majority of AMPs in current clinical development target skin infections caused by Gram-positive bacteria, i.e., topical use only [31]. In addition, within the last 30 years, only four natural AMPs have found their way onto the market and no systemic AMP has been approved by the Federal Drug Administration in the USA [31]. This dearth of clinically approved AMPs despite the past three decades of attempts and the excellent antimicrobial activity of many AMPs lies mainly in their generally high toxicity to normal cells which prevents their use as a systemic drug. Interestingly, cationic AMPs polymyxin B and polymyxin E (colistin) saw widespread use in the 1960s and 1970s. However, their clinical use in the 1970s was scaled back considerably due to serious neurotoxicity and nephrotoxicity issues [32][33][34][35][36]. Despite these toxicity drawbacks, these two peptides returned as antibiotics of last resort with the emergence of prevalent Gram-negative bacteria with multidrug resistance. However, the aforementioned emergence of polymyxin-resistant "Superbugs" [32,33,36], due to the fact that these particular peptide antibiotics (unlike the AMPs presently under consideration) have specific targets and are thus prone to resistance, means that it is now critical to develop antimicrobials effective against both polymyxin B-and colistin-resistant microorganisms. Worldwide research for the past 30 years to remove toxicity from AMPs, thus enabling a shift of focus from development of peptide drugs for topical use towards agents for systemic administration, has been unsuccessful until recent work in our laboratory.
Numerous structure/activity studies on both natural and synthetic AMPs identified factors important for antimicrobial activity: the presence of both hydrophobic and basic (positively charged) residues, an amphipathic nature, and preformed or inducible secondary structure (α-helix or β-sheet) [16]. We have always postulated that a de novo design synthetic peptide approach to examining the effect of incremental changes in these parameters would enable rapid progress in the rational design of novel peptide AMPs. Thus, from lessons learned about factors important for antimicrobial activity, as noted above, we utilized the structural framework, or template, of a 26-residue amphipathic α-helical AMP with excellent antimicrobial activity but with, initially, strong hemolytic activity [16]. The 26-residue length of the template was designed to be able to accept amino acid substitutions with minimal effects on peptide properties and stability other than the ones under investigation; at the same time, synthesis and purification of analogs remained straightforward. With this template approach, we determined the effect on biological activity of varying the hydrophobicity of the non-polar face [37] or the number of positively charged residues on the polar face [38]. In addition, utilizing D-enantiomers of amino acids led to excellent stability against proteolytic digestion (a key property for AMPs to be useful as injectable AMPs), whilst maintaining excellent antimicrobial activity [39].
At this point, a major milestone was our discovery of "specificity determinants" allowing selectivity between eukaryotic cells and Gram-negative microorganisms, i.e., producing a major decrease in toxicity as measured by hemolysis of human red blood cells [39][40][41]. These "specificity determinants" were one, later two, Lys-substitutions in the middle of the non-polar face of the amphipathic model peptide, a peptide long enough to allow such substitutions whilst maintaining sufficient hydrophobicity on the non-polar face. Briefly, we utilized positively charged residues as specificity determinants (Lys residues at positions 13 and 16 of the non-polar face) of the 26-residue peptide. In addition, we manipulated total hydrophobicity, hydrophobe type and location as design parameters. Taken together, these approaches resulted in unprecedented, at that time, improvements in therapeutic indices (hemolytic activity/antimicrobial activity) [40,41]. This discovery hastened another aspect of our template design, namely the requirement for our peptides to lie parallel to the membrane, surface, i.e., promoting the "carpet model" of interaction [24,42,43] while preventing penetration of the membrane as a transmembrane helix in eukaryotic cells via a "barrel stave" mechanism [24,44], thus preventing hemolysis. Further, we demonstrated that modification of native AMPs (the 22-residue Piscidin 1 and 28-residue Dermaseptin S4) with Lys specificity determinants in the non-polar face of these amphipathic α-helical peptides produced similar results of improved antimicrobial activity and dramatically decreased hemolytic activity [45,46]. Such results are of critical importance to the future of AMPs as therapeutic agents.
The present study serves to continue the success of our template-driven de novo design approach by attempting to fine-tune our recent achievement of utilizing unusual amino acids (Dab and Dap) in the polar face of our AMP to eliminate hemolysis [47]. Thus, we have determined the effect of changing locations of positively charged residues on the polar face of the AMP, as well as eliminated a single positively charged residue at the C-terminal which allows future development of Pegylated AMPs on a C-terminal cysteine residue if prolonged half-life is necessary.

Solid-phase peptide synthesis and reversed-phase purification
The synthesis and purification methods have been described in detail in a previous publication [47].

Characterization of helical structure
Circular dichroism (CD) spectroscopy was used to determine the mean residue molar ellipticities of the peptides, using a Jasco J-815 spectropolarimeter (Jasco, Inc., Easton, MD, USA) under two sets of conditions: at pH 7.0 the buffer was 50 mM NaH 2 PO 4 /NaHPO 4 /100 mM KCl and in the presence of an α-helix inducing solvent, 2, 2, 2-trifluoroethanol, TFE, (50 mM NaH 2 PO 4 /NaHPO 4 /100 mM KCl, pH 7.0 buffer/50% TFE). A 10-fold dilution of an approximately 500μM stock solution of the peptides was loaded into a 0.1 cm quartz cell and its ellipticity scanned from 195 to 250 nm. Peptide concentrations were determined by quantitative amino acid analysis.

Amino acid analysis for peptide quantitation
The method of Cohen and Michaud [55] was used for amino acid analysis. Each peptide sample was aliquoted into glass tubes and lyophilized followed by acid hydrolysis in 6 M HCl with 0.1% phenol for 48 h at 110°C. The resulting solution was allowed to come to room temperature and then vacuum-dried to remove the HCl. Each sample was then resuspended in 10 mM HCl and 20 μL of sample was added to 60 μL of 0.2M sodium borate buffer, pH 8.8. To this mixture, 20 μL of 6-aminoquinoyl-N-hydroxysuccinimidyl carbamate in acetonitrile was added to derivatize the amino acids present in the sample. This sample was then heated to 55°C for 15 min to convert Tyr byproducts to one form. An Agilent 1260 series instrument with a Waters AccQTag column, 3.9 mm I.D. × 150 mm column was used to separate and quantify the derivatized amino acids present in each sample using UV absorbance at 254 nm.

Gram-negative bacterial strains used in this study
The A. baumannii strains used in this study consisted of seven strains resistant to Polymyxin B and Colistin (antibiotics of last resort) obtained from MERCK (M89941, M89949, M89951, M89952, M89953, M89955 and M89963). The MIC GM in this case is the geometric mean MIC from the seven Acinetobacter baumannii strains used in this study.

Antimicrobial activity (MIC) determination
The minimal inhibitory concentration (MIC) is defined as the lowest peptide concentration that inhibited bacterial growth. MICs were measured by a standard microtiter dilution method in Mueller Hinton (MH) medium. Briefly, cells were grown overnight at 37°C in MH broth and were diluted in the same medium. Serial dilutions of the peptides were added to the microtiter plates in a volume of 50 μL, followed by the addition of 50 μL of bacteria to give a final inoculum of 5 × 10 5 colony-forming units (CFU)/mL. The plates were incubated at 37°C for 24h, and the MICs were determined. The MIC GM is the geometric mean of the number of MIC values.

Hemolytic activity (HC 50 ) determination
Peptide samples (concentrations determined by amino acid analysis) were added to 1% human erythrocytes in phosphate-buffered saline (100 mM NaCl, 80 mM Na 2 HPO 4 , 20 mM NaH 2 PO 4 , pH 7.4) and the reaction mixtures were incubated at 37°C for 18h in microtiter plates. Two-fold serial dilutions of the peptide samples were carried out. This determination was made by withdrawing aliquots from the hemolysis assays and removing unlysed erythrocytes by centrifugation (800 × g). Hemoglobin release was determined spectrophotometrically at 570 nm. The control for 100% hemolysis was a sample of erythrocytes treated with water. The control for no release of hemoglobin was a sample of 1% erythrocytes without any peptide added. Since erythrocytes were in an isotonic medium, no detectable release (<1% of that released upon complete hemolysis) of hemoglobin was observed from this control during the course of the assay. The hemolytic activity HC 50 is the peptide concentration that causes 50% hemolysis of erythrocytes after 18h. HC 50 was determined from a plot of percent lysis versus peptide concentration (μM) using 12 different concentrations up to 1000 micrograms per ml for 18h at 37°C. The average of 3 replicates is used with an average variance of less than 4%. Fresh human blood was obtained from Vitalant, Denver, CO, USA.

Therapeutic index (T.I.) determination
The therapeutic index is a widely accepted parameter to represent the specificity of antimicrobial peptides for prokaryotic versus eukaryotic cells. It is calculated by the ratio of hemolytic activity (HC 50 ) and antimicrobial activity (MIC GM ). The MIC GM in this case is the geometric mean MIC from the seven Acinetobacter baumannii strains used in this study.
Thus, larger values of therapeutic index indicate greater specificity for prokaryotic cells. Thus, the therapeutic index is the HC 50 /MIC GM ratio.

Results and Discussion
Peptide design, location and type of positively charged residue on the polar face In this study, we designed de novo, synthesized, purified and characterized ten potentially  (Table 1). All ten AMPs have two "specificity determinants" (D-Lys residues at 13 and 16 in the center of the non-polar face). We have previously shown the critical importance of "specificity determinants" in these AMPs which encoded selectivity for Gram-negative pathogens and removed both Gram-positive activity and hemolytic activity from broad spectrum AMPs [40,41,[45][46][47]. In addition, we have shown that specificity determinants have another important role of preventing high-affinity to human serum proteins [40,41,45,46].  (Table 1) was not expected to have any undesired effect on the conformation since our objective was to have as little α-helical structure as possible in aqueous conditions but maximum inducible α-helical structure in the presence of the hydrophobicity of the membrane (mimicked here by determining the helical structure by circular dichroism spectroscopy (CD) in the presence of 50% trifluoroethanol). We did not expect the 5 or 6 L-substitutions of Dab or Dap residues to affect the overall structure in any significant way since there are 20 or 21 positions out of 26 to maintain the structure in the presence of the hydrophobicity of the membrane. The use of the L-conformation for Dab and Dap residues was based on the fact that they are significantly less expensive for peptide synthesis and would not introduce any susceptibility to proteases since the Dab and Dap residues are unusual amino acids and are not recognized by proteases. The hydrophobic/non-polar faces of all ten AMPs have eight Leu residues in two clusters of four (colored yellow) separated by two Lys residues (specificity determinants in the center of the non-polar face (colored red)) ( Figure 2). Position 1 in all peptides is D-Lys which we consider is on the non-polar face; thus, the non-polar face contains three D-Lys residues at positions 1, 13 and 16 to give a net charge of +3 on the nonpolar face and +5 or +6 on the polar face resulting in an overall net charge on these AMPs of either +9 or +8 depending on whether there are 6 or 5 positively charged residues on the polar face (Table 1).
In the helical wheels, the non-polar face is indicated as a yellow arc (Leu residues are colored yellow and position Lys 1 and the specificity determinants at positions 13 and 16 are colored pink). The polar face is indicated as a black arc (positively charged residues are colored blue). In the helical nets, the residues on the non-polar face are circled with the Lys residues colored red (Lys 1 and the specificity determinants, Lys 13 and Lys 16) and the Leu residues in two clusters (L2, L5, L6, L9 for the N-terminal cluster and L17, L20, L21 and  Table 2). The MIC GM changed from 0.4 μM to 0.7 μM to 0.8 μM for D-Lys, L-Dab and L-Dap, respectively, when in the −2 location (compare D88, D89 and D106, Table 2). The largest difference in the two locations occurs when Dap residues are used (compare D105(Lys 1 -6 Dap-1) MIC GM of 1.2 μM with D106(Lys 1 -6 Dap-2) MIC GM of 0.8 μM). There seems to be a major advantage to have Dap residues at position 14 and 15 on the polar face rather than positions 11 and 18. Positions 14 and 15 are between the two specificity determinants (D-Lys residues) on the non-polar face at positions 13 and 16. This is creating a positively charged cluster in the sequence (D-Lys13, L-Dap14, L-Dap15 and D-Lys 16) ( Table 2). In the big picture, the changes in the geometric mean MIC value are minor compared to the effect observed in hemolytic activity by changing the residues on the polar face from D-Lys to L-Dab and L-Dap residues (4 carbon atoms, 2 carbon atoms and 1 carbon atom in the side-chain, respectively ( Table 2). We discovered that we can eliminate the positively charged residue at position 26 with no significant effect on the geometric mean MIC value (compare D86(Lys 1 -6 Dab-1), MIC GM 0.9 μM to D102(Lys 1 -5 Dab-1), MIC GM 0.7 μM and D89(Lys 1 -6 Dab-2), MIC GM 0.7 μM to D104(Lys 1 -5 Dab-2), MIC GM 0.8 μM) ( Table 2).

Hemolytic activity and therapeutic indices
The biological activities of the ten peptide analogs are shown in table 2. The hemolytic activity is expressed as the HC 50 value which is the concentration of peptide that results in 50% hemolysis of human red blood cells. In order to determine that we were able to eliminate hemolysis of human red blood cells, we used the most rigorous test of hemolytic activity (18h at 37°C and up to 1000 μg/mL or >350 μM of AMP). This is in stark contrast to other researchers who routinely use incubation times of just 0.5-2h. We have shown that, when the exposure time is increased from less than 2 h to 18h [16,20,39], substantially greater hemolysis is observed. Clearly, hemolysis should be monitored on human red blood cells for an exposure time up to 18h, since anything less will lead to misleading results.

Peptide hydrophobicity
Retention behavior in reversed-phase high-performance liquid chromatography (RP-HPLC) is an excellent method to represent overall peptide hydrophobicity. Even though the nonpolar face of an amphipathic α-helical peptide represents the preferred binding domain for its interaction with the hydrophobic matrix of the reversed-phase column [56,57]; the overall hydrophobicity is also affected by the composition of residues on the polar face (five or six positively charged residues) (Figure 1). The RP-HPLC results for these two series of peptides are shown in figure 4

Peptide helicity
The biophysical data for our ten peptides are shown in table 3. Circular dichroism (CD) spectroscopy was used to determine the α-helical content in aqueous conditions at pH 7 (50 mM PO 4 , 100 mM KCl) and in the presence of 50% trifluoroethanol (TFE) to mimic the hydrophobicity and α-helix inducing ability of the hydrophobic membrane, the target of our AMPs. Our strategy was to minimize α-helical structure in aqueous conditions and maximize the inducible α-helical structure in the presence of the hydrophobicity of the membrane. The % helix in aqueous conditions varied from 6 to 29% and the % inducible α-helix varied from 71 to 94% depending on the peptide ( Table 3). The specificity determinants (Lys residues at positions 13 and 16 in the center of the non-polar face) were used to disrupt the continuous hydrophobic face of our template, creating two hydrophobic clusters of leucine residues, cluster one consisted of leucine residues at positions 2, 5, 6 and 9 and cluster two consisted of leucine residues at positions 17, 20, 21 and 24 ( Figure  2). Though all AMPs met the general requirement of low α-helical content in aqueous conditions and dramatic increases in α-helical content in the presence of 50% TFE, there was no direct correlation with the type of positively charged residue (Lys, Dab or Dap) used on the polar face and helical content. In summary, inducible α-helical structure plays a critical role in providing our AMPs with desired properties.

Conclusion
The goal of the present study was to determine whether our template-driven de novo designed peptide approach which enabled us to fulfill the long-sought goal of eliminating toxicity from AMPs could be further refined to improve therapeutic indices even more, as well as allow pegylation of the peptide model to enhance Interestingly, the location of positively charged residues on the polar face had a major effect on hemolysis and the best location was dependent on whether Dab or Dap residues were used, i.e., side-chain length, number of carbon atoms and residue location all appear to affect hemolysis. Significantly, the therapeutic index of the 6Dab-containing −1 analog (>1012) rose to >1589 for the 6Dab-2 analog, an impressive increase in efficacy. In addition, the 5Dab-1 analog with a T.I. of >1012 saw an even greater increase in T.I. (>1863) for the 5Dab-2 peptide.
Comparing the 6Dab and 5Dab peptide series, the T.I. values for 6Dab-1 and 5Dab-1 were >824 and >1012, respectively; for the 6Dab-2 and 5Dab-2 peptides, the T.I. values were >1589 and >1863, respectively. Thus, we have shown that we can remove the C-terminal positively charged residue and replace it with Ser26 without any consequence; indeed, for the Dab analogs, an improvement in T.I. was observed. Such results now allow us to investigate the effectiveness of pegylation to a C-terminal Cys residue, in place of a positively charged residue, in order to prolong peptide half-life when desired.
Our continuing studies clearly show the potential of our amphipathic AMPs as potential therapeutics to replace existing antibiotics as well as the leading edge peptide design which our de novo designed template represents.

HC 50
Peptide concentration that results in 50% lysis of human red blood cells (hemolytic activity)

MIC GM
Minimal inhibitory concentration geometric mean of the number of strains tested
In the helical wheels, the non-polar face is indicated as a yellow arc (Leu residues are colored yellow and position Lys 1 and the specificity determinants at positions 13 and 16 are colored pink). The polar face is indicated as a black arc (positively charged residues are denoted by X and are colored blue).    Table 1).  The helical content (as a percentage) of a peptide is relative to the molar ellipticity value of the peptide in the presence of 50% TFE. % helix induced is the increase in molar ellipticity (as a percentage) of the peptide in the presence of 50% TFE.